The need for human organs far outstrips the supply. One potential transformative solution is to humanize the immune repertoire of pig organs in order to limit patient rejection of xenotransplanted organs. Whole genes and pathways can in principle be put into pig cells using a large artificial chromosome, which the Boeke lab refers to as synthetic Pig Artificial Chromosomes (sPACs). Current versions of these mammalian artificial chromosomes have limitations. The lab will address these issues by completely redesigning the sPACs from the `bottom up' in the yeast Saccharomyces cerevisiae. These yeast-based sPACs could be readily reprogrammed with patient data and quickly uploaded into pig cells. In order to achieve this `plug and play' capability, the primary goal of this postdoctoral proposal is to humanize the chromatin of S. cerevisiae, thus ensuring the generation of mammalian-like chromosome structure. The proposal will focus on three specific aims: 1) humanize the histones of yeast; 2) build in H3K27 methylation; and 3) build in H3K9 methylation. In addition, the proposal will permit the study of the de novo establishment of mammalian chromatin in a cell. This will lead to fundamental insight into methylation establishment, maintenance, and mechanisms of perturbation, information relevant to human gene expression. This proposal will achieve two innovative and complementary goals by building human chromatin into yeast: study the mechanisms of chromatin establishment in a cellular 'blank slate', and generate a cell capable of making pig artificial chromosomes designed to facilitate organ xenotransplantation. The long-term goal is to provide an unlimited method of generating humanized pig organs for organ transplantation, thus advancing the treatment of human disease.